Enhancer RNAs represent an exciting new class of transcripts that arise from gene expression enhancers across the genome. These enhancer derived RNAs mark active enhancers and the regulatory regions of the genome that are responsible for driving gene expression programs. Due to technical limitations, enhancers RNAs have not been thoroughly explored in a clinical setting. Accurate quantification of enhancer RNAs could transform cancer diagnostics by providing a high-resolution map of gene regulation events across the genome. In particular, drugs that target epigenetic modifying enzymes, which have shown great promise across many different cancer types, should impact gene regulation on a genome-wide scale. Analysis of enhancer RNAs will provide a direct readout of enhancer activity and interrogate the main mechanism of action of epigenetic modifying drugs. To explore the clinical utility of enhancer RNA quantification we propose to develop an approach, called FFPEcap-seq, which will enable the accurate genome-wide measurement of enhancer RNAs in the most commonly available form of patient samples, formalin-fixed paraffin embedded (FFPE). This project outlines the development of FFPEcap-seq in three phases: (1) Optimization in freshly collected RNA, (2) Testing in FFPE derived RNA, and (3) Application to HDAC inhibitor treated cell lines and patient samples. In phase 1, we will optimize FFPEcap-seq on fully intact RNA using defined quality control metrics. For phase 2, we will collect RNA from matched freshly grown cells and FFPE cells. This powerful resource will allow for the comparison of FFPEcap-seq and other methods (not originally designed for FFPE RNA), in both fully intact and FFPE degraded RNA from the same sample. During phase 3, FFPEcap-seq will be applied to cell lines and patient biopsies pre- and post-treatment with HDAC inhibitors, an exciting class of epigenetic modifying drugs. The overall goal of this project is to produce a method that will permit the analysis of enhancer RNAs in FFPE patient samples as well as showcase FFPEcap-seq in pilot studies with HDAC inhibitor treated samples. Over the long term, FFPEcap-seq could find wide adoption as a technique for both patient selection/monitoring and discovering genomic loci that are responsible for aberrant gene regulation observed in many cancer types.